The re-emergence and global spread of mosquito-borne diseases is reducing the health of human and wildlife populations worldwide, and is an increasing drain on the world economy. In particular, mosquito borne viruses are re-emerging in endemic areas and rapidly spreading into new ranges across the globe (eg. West Nile virus in the Americas, Rift Valley fever virus in the Middle East, O'nyong-nyong virus in Africa). As a fundamental process for transmission, these arboviruses must replicate in mosquito gut tissues, disseminate throughout the mosquito body, and eventually infect the mosquito salivary glands. Using alphavirus transducing systems that express green fluorescent protein, we have been able to characterize routes of arboviral dissemination in mosquitoes. We have also made use of currently available genome data for Anopheles gambiae and Aedes aegypti mosquitoes, and with these data are beginning to characterize barriers to dissemination in vector mosquitoes. Through these studies, we are understanding that mosquitoes have natural innate immune defenses against arboviral infections that are primarily driven by RNA interference. At the same time, we are recognizing that some arboviruses have evolved mechanisms to avoid this innate immunity. It is critical that we characterize this interplay between mosquito anti-viral immunity and arboviruses to better design control methods for mosquito-borne disease. In this proposal, we focus our studies on vector mosquitoes that are responsible for the bulk of mosquito-borne disease in human populations. The specific aims of this proposal are as follows: 1) Define the barriers to O'nyong-nyong virus dissemination in Anopheles gambiae mosquitoes. 2) Characterize the infection and dissemination barriers ;or Sindbis viruses in Aedes aegypti mosquitoes and define these barriers in the context of two different Sindbis virus strains. 3) Characterize the mechanisms that Culex tritaeniorhynchus mosquitoes use to clear Sindbis virus-infected midgut cells. [unreadable] [unreadable]